CN114317688A - Visual mixed dye and application thereof in LAMP detection - Google Patents
Visual mixed dye and application thereof in LAMP detection Download PDFInfo
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- CN114317688A CN114317688A CN202210101686.8A CN202210101686A CN114317688A CN 114317688 A CN114317688 A CN 114317688A CN 202210101686 A CN202210101686 A CN 202210101686A CN 114317688 A CN114317688 A CN 114317688A
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- 230000000007 visual effect Effects 0.000 title claims abstract description 28
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 238000006243 chemical reaction Methods 0.000 claims abstract description 50
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 41
- AUIINJJXRXMPGT-UHFFFAOYSA-K trisodium 3-hydroxy-4-[(2-hydroxy-4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2,7-disulfonate Chemical compound [Na+].[Na+].[Na+].Oc1cc(c2ccccc2c1N=Nc1c(O)c(cc2cc(ccc12)S([O-])(=O)=O)S([O-])(=O)=O)S([O-])(=O)=O AUIINJJXRXMPGT-UHFFFAOYSA-K 0.000 claims abstract description 15
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229960002378 oftasceine Drugs 0.000 claims abstract description 13
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims abstract description 10
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims abstract description 10
- 229940099607 manganese chloride Drugs 0.000 claims abstract description 10
- 235000002867 manganese chloride Nutrition 0.000 claims abstract description 10
- 239000011565 manganese chloride Substances 0.000 claims abstract description 10
- 230000003321 amplification Effects 0.000 claims description 19
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 235000012736 patent blue V Nutrition 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 abstract description 6
- 150000007523 nucleic acids Chemical class 0.000 abstract description 6
- 102000039446 nucleic acids Human genes 0.000 abstract description 6
- 238000007397 LAMP assay Methods 0.000 description 43
- 239000000975 dye Substances 0.000 description 34
- 239000000243 solution Substances 0.000 description 7
- 239000000843 powder Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 101710163270 Nuclease Proteins 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000011777 magnesium Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000011901 isothermal amplification Methods 0.000 description 2
- 229910001425 magnesium ion Inorganic materials 0.000 description 2
- 239000011572 manganese Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- XIAYFENBYCWHGY-UHFFFAOYSA-N 2-[2,7-bis[[bis(carboxymethyl)amino]methyl]-3-hydroxy-6-oxoxanthen-9-yl]benzoic acid Chemical compound C=12C=C(CN(CC(O)=O)CC(O)=O)C(=O)C=C2OC=2C=C(O)C(CN(CC(O)=O)CC(=O)O)=CC=2C=1C1=CC=CC=C1C(O)=O XIAYFENBYCWHGY-UHFFFAOYSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003269 fluorescent indicator Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- -1 pyrophosphate ions Chemical class 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
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Abstract
The invention discloses a visual mixed dye and application thereof in LAMP detection, wherein the visual mixed dye comprises a reagent A and a reagent B, and the volume ratio of the reagent A to the reagent B is 1: 1-2; the reagent A contains calcein and manganese chloride, wherein the concentration of the calcein is 500 mu M, and the concentration of the manganese chloride is 10 mM; the reagent B contains hydroxynaphthol blue, and the concentration of the hydroxynaphthol blue is 0.32 mM. Compared with the conventional common visual dye, the visual mixed dye is used for LAMP detection, so that the LAMP reaction result is easier to judge, the color difference between a negative result and a positive result is more obvious, the judgment of the result is avoided to a great extent, and complicated instrument equipment is not needed for assisting in detecting the experimental result; in addition, the visual mixed dye is added into the LAMP reaction system in advance, and a PCR tube cover does not need to be opened after the LAMP reaction, so that the nucleic acid pollution to the laboratory environment is avoided.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a visual mixed dye and application thereof in LAMP detection.
Background
Loop-mediated isothermal amplification (LAMP) is a nucleic acid isothermal amplification technology that has been developed rapidly in recent years. The kit can amplify a target gene sequence at a constant temperature so as to realize the rapid detection of a target gene, and has attracted more and more attention due to the advantages of short time, high sensitivity, good specificity, convenient operation and the like. At present, reading of LAMP results is mainly divided into 3 types of methods such as turbidity detection, agarose gel electrophoresis detection, fluorescence/visualization dye detection and the like. However, since the turbidity detection and the electrophoresis detection both depend on instruments and require complicated operations, the visual dye detection method is more and more favored by experts. According to the method, only the dye is added into the reaction system before the LAMP reaction is started, and after the reaction, the negative reaction reagent and the positive reaction reagent show obvious color difference, so that the reaction result is quickly interpreted.
Currently, the dyes used more widely are composed of two types: 1) hydroxynaphthol Blue (HNB) as metal ion indicator, deep purple powder, easily soluble in water and ethanol, or soluble in water and ethanolAs a visual indicator of LAMP reaction. Adding HNB aqueous solution in advance in the LAMP system, initially combining magnesium ions with HNB to enable reaction liquid to be purple, and Mg along with the reaction2+With precipitated P2O7 4-Reaction to form Mg2P2O7Precipitation, loss of Mg from HNB2+The color of the system is changed into sky blue, and the unreacted system still keeps violet; 2) calcein (Calcein), bright yellow powder, ethanol and alkali soluble, slightly water soluble, fluorescent indicator, and Mg2+The combined compound can generate strong fluorescence, and negative and positive results under natural light have certain difference but are not obvious. Firstly, Mn is added into calcein2+，Mn2+The green fluorescence of calcein was quenched. After LAMP amplification reaction occurs, pyrophosphate ions and Mn which are byproducts are generated2+Binding and releasing calcein, and relieving quenching state. The final result is that under the excitation of 365nm blue light, the positive reaction emits strong green fluorescence, and the negative reaction emits weaker green fluorescence.
The two dyes are usually used separately, but they have a great disadvantage that the difference in color between the negative and positive results is not obvious enough, or the result is read with the aid of an instrument, and the process is complicated. Especially under natural light, when the concentration of the target gene is too low, the weak positive result and the negative result are difficult to distinguish, so that the result interpretation is inaccurate.
Disclosure of Invention
In order to solve the technical problems, the invention provides a visual mixed dye and application thereof in LAMP detection.
The technical scheme adopted by the invention is as follows: a visual mixed dye comprises a reagent A and a reagent B, wherein the volume ratio of the reagent A to the reagent B is 1: 2;
the reagent A contains calcein and manganese chloride, wherein the concentration of the calcein is 500 mu M, and the concentration of the manganese chloride is 10 mM;
the reagent B contains hydroxynaphthol blue, and the concentration of the hydroxynaphthol blue is 0.32 mM.
Preferably, agent a and/or agent B is prepared from enucleated enzyme water.
The application of the visual mixed dye in LAMP detection.
Preferably, the reaction mixture is added to the LAMP reaction system in advance before the LAMP reaction.
Preferably, before LAMP amplification, the reaction system is grayish blue;
after LAMP amplification is finished, a positive reaction system is sky blue, and a negative reaction system is violet.
The invention has the advantages and positive effects that: compared with the common visual dye at present, the visual mixed dye disclosed by the scheme enables the LAMP reaction result to be easier to judge, the color difference between the negative result and the positive result is more obvious, the judgment of the result is greatly avoided, and complex instruments and equipment are not needed for assisting in detecting the experimental result;
in addition, the visual mixed dye is added into an LAMP reaction system in advance, and a PCR tube cover does not need to be opened after LAMP reaction, so that the nucleic acid pollution to the laboratory environment is avoided;
the visualized mixed dye is a non-DNA-embedded dye, has small interference on LAMP reaction, and does not influence the sensitivity and specificity of LAMP detection.
Drawings
FIG. 1 is the development of a novel mixed dye prior to LAMP amplification in an embodiment of the present invention;
FIG. 2 shows the color development of the novel mixed dye after LAMP amplification in the embodiment of the present invention;
FIG. 3 is an electrophoretogram of nucleic acids according to an embodiment of the present invention;
FIG. 4 shows the color development before LAMP amplification in the comparative example of the present invention;
FIG. 5 shows the color development of the comparative example of the present invention after LAMP amplification;
1：106copies/μL，2：105copies/μL，3：104copies/μL，4：103copies/μL，5：102copies/μL，6：101copies/μL，7：1copies/μL，8：NTC。
Detailed Description
Embodiments of the present invention will be described below with reference to the accompanying drawings.
The invention discloses a visual mixed dye, which comprises a reagent A and a reagent B, wherein the volume ratio of the reagent A to the reagent B is 1: 2; the reagent A contains calcein and manganese chloride, and is dissolved by nuclease water, the concentration of the calcein is 500 mu M, and the concentration of the manganese chloride is 10 mM; the reagent B contains hydroxynaphthol blue, and is dissolved by using nuclease water, and the concentration of the hydroxynaphthol blue is 0.32 mM. The visual mixed dye can be applied to LAMP detection, is added into an LAMP reaction system before LAMP reaction, and after the reaction is finished, the positive and negative can be directly judged according to the system color, and the color contrast difference is obvious; before LAMP amplification, a reaction system is grayish blue; after LAMP amplification is finished, a positive reaction system is sky blue, and a negative reaction system is violet.
The following describes the scheme of the present invention with reference to the accompanying drawings, wherein experimental methods without specific description of operation steps are all performed according to corresponding commercial specifications, and instruments, reagents and consumables used in the examples can be purchased from commercial companies without specific description.
Example 1:
1.1 preparation of visual Mixed dyes
Weighing calcein powder and manganese chloride powder, placing in the same container, dissolving with nuclease, adjusting pH to alkalescence to obtain reagent A, and storing at 4 deg.C.
Weighing HNB powder, dissolving with nuclease water to make the concentration of HNB in the solution be 0.32mM, fully and uniformly mixing, and the obtained solution is a reagent B, namely single HNB dye storage solution, and storing at 4 ℃ for later use.
And (3) fully and uniformly mixing the reagent A and the reagent B according to the volume of 1:2 to obtain a solution, namely the visual mixed dye, and storing the solution at 4 ℃ for later use.
1.2 visual Mixed dye for LAMP amplification reaction
The LAMP reaction system was configured as follows.
KPC signature sequence:
CTGACCAACCTCGTCGCGGAACCATTCGCTAAACTCGAACAGGACTTTGGCGGCTCCATCGGTGTGTACGCGATGGATACCGGCTCAGGCGCAACTGTAAGTTACCGCGCTGAGGAGCGCTTCCCACTGTGCAGCTCATTCAAGGGCTTTCTTGCTGCCGCTGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTTACGGCAAAAATGCGCTGGTT
designing a KPC-LAMP primer group according to the KPC characteristic sequence, wherein the information is as follows:
F3-KPC：TCGAACAGGACTTTGGCG
B3-KPC：AACCAGCGCATTTTTGCC
FIP-KPC：CCTCAGCGCGGTAACTTACAGTTTTTCTCCATCGGTGTGTACGC
BIP-KPC：TCAAGGGCTTTCTTGCTGCCGTTTTACGGATGGGTGTGTCCAG
LF-KPC：GCCTGAGCCGGTATCCAT
LB-KPC：CCAGCAGCAGGCCGGCTT
taking a positive control strain of KPC gene as a positive control strain, taking a DNA template extracted from the positive control strain as initial concentration, and adjusting the initial concentration to 106CFU/mu L, then performing 10-fold serial gradient dilution, naming the reaction tube as No. 1-8 tube, adding KPC-DNA templates with different concentrations into LAMP amplification reaction system in No. 1-7 tube in sequence, the concentration is 1 × 106CFU，1×105CFU，1×104CFU，1×103CFU，1×102CFU，1×101CFU，1×100A CFU; tube 8 is a negative control NTC tube, and the same volume of enucleated enzyme water is added.
The prepared LAMP reaction system is placed at the constant temperature of 65 ℃ for reaction for 45min, and then placed at the temperature of 80 ℃ for 10min to terminate the reaction. And (4) judging the difference between the positive result and the negative result after LAMP amplification by visual observation.
And (4) interpretation of results: under natural light, the color change of the reaction solution is observed by naked eyes, the result is shown in figure 1, and before LAMP amplification, positive and negative reaction systems containing visual mixed dyes are all grey blue; after the LAMP amplification is finished, the result is shown in figure 2, the positive reaction system containing the visual mixed dye is sky blue, and the negative reaction system is violet; all results were consistent with the nucleic acid electropherogram results of FIG. 3. According to the judgment of naked eyes, the color difference between the positive result and the negative result of the reagent containing the novel mixed dye can be clearly seen, and the judgment of the visual result is easy.
In addition, the more the content of the DNA template in the system is, the darker the color is, and the DNA content in the two samples can be directly compared by a direct colorimetric method.
Compared with the common dyes at present, the visualized mixed dye enables LAMP results to be easier to interpret, the color difference between positive results and negative results is more obvious, the misinterpretation of the results is greatly avoided, and complex instruments and equipment are not needed to assist in detecting the experimental results; in addition, the dye is added into the LAMP amplification reaction solution in advance, and a PCR tube cover does not need to be opened after the LAMP reaction, so that the nucleic acid pollution to the laboratory environment is avoided; finally, the visualized mixed dye is a non-DNA-embedded dye, has small interference on LAMP reaction, and does not influence the sensitivity of LAMP detection.
Comparative example:
in the prior art, the patent disclosed in CN108315390A also discloses a mixed dye comprising a reagent a and a reagent B, wherein the reagent a contains calcein and manganese chloride, and the reagent B contains hydroxynaphthol blue. However, the mixing ratio of the reagent A and the reagent B in the patent is different, according to the mixing ratio range 1.8-2.2:1 disclosed in the patent, the mixing ratio is 2:1, the mixed dye is prepared and is also used in the LAMP amplification reaction system in the example 1, the results before and after amplification are shown in FIGS. 4 and 5, after the isothermal amplification at 65 ℃, the color difference between the negative control and the positive control is not obvious, and the distinguishing difficulty coefficient is large.
The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Sequence listing
<110> hematological disease Hospital of Chinese medical science (institute of hematology of Chinese medical science)
TIANJIN XINUO BIOLOGICAL PHARMACEUTICAL Co.,Ltd.
TIANJIN ERA BIOLOGY TECHNOLOGY Co.,Ltd.
<120> visual mixed dye and application thereof in LAMP detection
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ttcccactgt gcagctcatt caagggcttt cttgctgccg ctgtgctggc tcgcagccag 180
cagcaggccg gcttgctgga cacacccatc cgttacggca aaaatgcgct ggtt 234
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tcgaacagga ctttggcg 18
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aaccagcgca tttttgcc 18
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cctcagcgcg gtaacttaca gtttttctcc atcggtgtgt acgc 44
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<213> Artificial Sequence (Artificial Sequence)
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tcaagggctt tcttgctgcc gttttacgga tgggtgtgtc cag 43
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gcctgagccg gtatccat 18
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ccagcagcag gccggctt 18
Claims (5)
1. A visual mixed dye, which is characterized in that: the reagent kit comprises a reagent A and a reagent B, wherein the volume ratio of the reagent A to the reagent B is 1: 2;
the reagent A contains calcein and manganese chloride, wherein the concentration of the calcein is 500 mu M, and the concentration of the manganese chloride is 10 mM;
the reagent B contains hydroxynaphthol blue, and the concentration of the hydroxynaphthol blue is 0.32 mM.
2. The visual mixed dye according to claim 1, wherein: the reagent A and/or the reagent B is prepared from enucleated enzyme water.
3. Use of the visualized mixed dye according to claim 1 or 2 in LAMP detection.
4. The use of the visualized mixed dye according to claim 3 in LAMP detection, characterized in that: is added into the LAMP reaction system in advance before the LAMP reaction.
5. The use of the visualized mixed dye according to claim 3 in LAMP detection, characterized in that: before LAMP amplification, a reaction system is grayish blue;
after LAMP amplification is finished, a positive reaction system is sky blue, and a negative reaction system is violet.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967790A (en) * | 2017-02-20 | 2017-07-21 | 浙江大学 | A kind of detection method of nucleic acid amplification |
| CN107022624A (en) * | 2017-05-08 | 2017-08-08 | 浙江农林大学 | The LAMP methods and kit of quick detection bakanae disease of rice germ from seed rice |
| CN108315390A (en) * | 2018-03-15 | 2018-07-24 | 吉林大学 | Visualization mixed dye for LAMP detections |
| CN108588183A (en) * | 2018-05-03 | 2018-09-28 | 佛山科学技术学院 | A kind of detection reaction system of calcein visualization LAMP detection Klebsiella Pneumoniaes |
-
2022
- 2022-01-27 CN CN202210101686.8A patent/CN114317688A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967790A (en) * | 2017-02-20 | 2017-07-21 | 浙江大学 | A kind of detection method of nucleic acid amplification |
| CN107022624A (en) * | 2017-05-08 | 2017-08-08 | 浙江农林大学 | The LAMP methods and kit of quick detection bakanae disease of rice germ from seed rice |
| CN108315390A (en) * | 2018-03-15 | 2018-07-24 | 吉林大学 | Visualization mixed dye for LAMP detections |
| CN108588183A (en) * | 2018-05-03 | 2018-09-28 | 佛山科学技术学院 | A kind of detection reaction system of calcein visualization LAMP detection Klebsiella Pneumoniaes |
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